Test 1 - Cultivation of Viruses (4)

Question 1

Why is it that viruses do not replicate on non-living media?

Answer 1

They do not have the genetic capability to multiply by division. Outside of the host cell, they are dormant particles. They need a living host cell to replicate by using the host machinery to produce proteins, nucleic acids for the next generation.

Question 2

What are the main ways to cultivate viruses?

Answer 2

1. Cell/tissue culture
2. Inoculate an embryonated egg
3. Laboratory animals

Question 3

Briefly describe the main concept of an organ culture.

Answer 3

This is when part of an organ or a whole organ is cultured in-vitro. It is a difficult technique that may have issues when done.

Question 4

Define a cell culture and the two main methods.

Answer 4

• Refers to the removal of cells from an animal or plant and their subsequent growin in a favorable artificial environment.
• The cells are grown in-vivo as a suspension culture or as a monolayer culture.

Question 5

What is the difference between a suspension culture and a monolayer culture?

Answer 5

• A suspension culture is used when cells do not require attachment for growth or attach to the vessel walls.
• A monolayer culture is when the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell thick.

Question 6

What is a primary cell culture?

Answer 6

This is the growth of cells dissociated directly from the parental tissue (e.g. kidney/liver) of human or animal origin.

They are morphologically similar to the parent tissue and have the same chromosomes as the original tissue.

Question 7

What are the advantages and disadvantages of using a primary cell culture?

Answer 7

Advantages

• Best culture for isolation and propagation of viruses
• Heterogenous - many cell types
• Closest to animal in similarity
• Used in producing viral vaccines

Disadvantages

• Difficult to obtain
• Relatively short lifespan (5-20 subcultures only)
• Very susceptible to contamination
• May not fully act like parent tissue due to complexity of culture media
  *

Question 8

What is a secondary/transfer culture?

Answer 8

This is when a primary culture is sub-cultured. It is the transfer of cels from one culture vessel to another. It is periodically required to provide fresh nutrients and growing space for continuously growing cell lines.

Question 9

What enzyme is primarily used to digest tissue/detach cells?

Answer 9

The proteolytic enzyme trypsin

Question 10

What is a cell line?

What are the types and how are they categorized?

Answer 10

This is after a primary culure has become sub-cultured.

It is categorized on the basis of the life span of the culture. There are two types:

1. Finite/Diploid cell lines: have a limited life span and go through a limited number of cell generations.
2. Continuous cell lines acquire the ability to divide indefinitely.

Question 11

Compare and contrast finite/diploid cell lines to continous/heteroploid cell lines.

Answer 11

Question 12

What are the different morphological categories? Which corresponds to each image?

Answer 12

1. Fibroblastic (or fibroblast-like) cells: bipolar or multipolar, have elongated shapes, grow attached to a substrate.
2. Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
3. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface (floating)

Question 13

What is the purpose of using culture medium?

What are some examples?

Answer 13

To provide all the necessary nutrients (amino acids, inorganic salts, vitamins and glucose) requried for growth of cells.

Eagle’s basal medium, leibovitz L-15 Medium

Question 14

What are some characteristics of serum that make it beneficial to use in culture media?

Answer 14

• It is a vital source of adhesion factors, attachment and spreading factors, low molecular weight nutrients and hormones and growth factors.
• Carrier proteins for lipoid substances and trace elements into the cell
• regulate cell membrane permeability

Question 15

What is the most widely used animal serum supplement?

Answer 15

Fetal bovine serum (FBS)

Question 16

TRUE/FALSE.

Growth medium has a higher percentage of serum than maintenance medium.

Answer 16

TRUE.

Growth medium = 5-10% serum

Maintenance medium = 0-2% serum

Question 17

What is the purpose of using a phenol red pH indicator?

Answer 17

This alerts you of contamination/changes in pH which may kill the cells.

Excretion may change pH.

Red (basic) = pH>7

Orange/yellow (acidic) = pH<7

Question 18

What kind of buffer is used to control pH changes?

Answer 18

Organic (e.g. HEPEPS) or CO2-bicarbonate based buffer (necessary to use exogenous CO2)

Question 19

What is added to medium to prevent contamination with bacteria, mycoplasma, yeast, molds, etc?

Answer 19

Antimicrobial agents

Question 20

TRUE/FALSE.

All cell types may be incubated at the same temperature.

Answer 20

FALSE.

Different species have different temperature requirements.

Question 21

What can you add in addition to trypsin to dissociate/detach cells

Answer 21

EDTA - it has an affinity for calcium, which inhibits trypsin.

Question 22

What are the two main proteases?

Answer 22

Trypsin and collagenase

Question 23

What is one method used by researchers to maintain the integrity of cells?

Answer 23

Using enzyme free dissociateion buffers. Incubating cell with too high trypsin concentration for too long may damage cell membranes and kill the cells.

Question 24

________ refers to damage or morphological changes to host cells during virus invasion.

Answer 24

Cytopathic effect or cytopathogenic effect (CPE)

Question 25

Describe the shell vial technique.

Answer 25

This is when there is a monolayer of viable cells on a coverslip. The coverslip is inserted culture medium and inoculated with specimen. It is then centrifuged, incubated and stained with anti-viral fluorescent monoclonal antibodies. Then, it is read with a fluorescent microscope.

Question 26

What are co-cultivated cells?

Answer 26

Single monolayer consisting of multiple [different] cell lines. It is ideal for the isolation of multiple viruses. Used to detect viral antigens by using a pool of luoresein-labeled monoclonal antibodies.

Question 27

What kind of eggs are used for viral innoculation?

Answer 27

Specific-pathogen-free (SPF) eggs

Question 28

What are the different routes of inoculation?

Answer 28

• Yolk Sac Inoculation
• Amniotic cavity Inoculation
• Allantoic Cavity Inoculation
• Chorioallantoic Membrane Inoculation

Question 29

What are some signs of viral growth in an engg inoculation?

Answer 29

1. Death of the embryo
2. Paralysis [sluggish movement]
3. Stunted growth
4. Urate deposits in the mesonephros
5. Hemorrhage and congestion
6. Hemagglutins in embryonic fluids
7. Extracellular membrane lesions
8. Pock (necrotic foci) on chorioallantoic membrane formed by vaccinia virus

Question 30

What are the methods of laboratory animal (mouse) inoculation?

Answer 30

Intracerebral

Intraperitoneal